The intestinal epithelium presents a barrier to invasion of the body by undesirable luminal contents. Using both immunohistochemistry and a new in vivo method for confocal and two-photon microscopy that allows study of living villi, we find that the intestinal epithelium is punctuated by discontinuities caused by cell shedding and potentially imperfect cell packing. Surprisingly, these cell-free gaps are filled with an impermeable substance that maintains the epithelial barrier. Our results suggest that the epithelium can seal the epithelial barrier prior to use of cell migration to heal gaps in the cell layer. While cell restitution will ultimately restore epithelial continuity, our hypothesis is that the mechanism we have observed is the major defender of barrier function immediately following villus cell shedding, and that perturbations of this mechanism lead to disease. Our goal in this R21 grant application is to address what we believe will be the fundamental points that will confirm or deny the importance of our findings prior to suggesting the project is worthy of full RO1 level funding. We will perform functional studies in normal and mutant mice using in vivo confocal and two-photon microscopy, and use immunohistochemistry and confocal endoscopy for comparative studies between mouse and human samples. In the first aim we will question the mechanisms that maintain barrier function during and after cell shedding. Specifically, we will use enzymatic digestion, immunohistochemistry and micro-analytical tools to define what class of material seals the gaps and sustains the epithelial barrier. We will also test if activation of myosin light chain kinase mediates a purse string closure that heals gaps. In the second aim we will ask where cells are lost and gaps are generated along a villus, using chimeric mice with a mosaic of crypt expression of EGFP to measure the fate of the cohort of cells from an individual crypt that migrate onto a living villus. The third aim is an initial confocal endoscopy study evaluating the characteristics, locale and frequency of epithelial gaps in non-diseased human ileal tissue. The outcome of these studies will define molecules involved in sealing and healing of gaps, and set the stage for us to define the importance of gaps in healthy and diseased tissue (in particular diseases having altered intestinal permeability) in later work. Last year we discovered a new way for the intestinal lining to provide a barrier that protects the body from luminal contents, in which the body is able to temporarily fill a gap in the cell layer lining the gut with an impermeable substance, instead of immediately using another (new) cell to plug the gap. Using advanced microscopy methods that let us study living tissues in mouse and human intestine, we will work to understand the underlying mechanism of gap sealing in mouse, and evaluate the sites and frequency of the gaps in human intestine. The outcomes of these studies will poise us to evaluate the importance of gaps in human disease as a next step.